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Magnetic resonance spectroscopy (MRS) methods offer a potentially valuable window into cellular metabolism. Measurement of flux between inorganic phosphate (Pi) and ATP using (31)P MRS magnetization transfer has been used in resting muscle to assess what is claimed to be mitochondrial ATP synthesis and has been particularly popular in the study of insulin effects and insulin resistance. However, the measured Pi→ATP flux in resting skeletal muscle is far higher than the true rate of oxidative ATP synthesis, being dominated by a glycolytically mediated Pi↔ATP exchange reaction that is unrelated to mitochondrial function. Furthermore, even if measured accurately, the ATP production rate in resting muscle has no simple relationship to mitochondrial capacity as measured either ex vivo or in vivo. We summarize the published measurements of Pi→ATP flux, concentrating on work relevant to diabetes and insulin, relate it to current understanding of the physiology of mitochondrial ATP synthesis and glycolytic Pi↔ATP exchange, and discuss some possible implications of recently reported correlations between Pi→ATP flux and other physiological measures. [ABSTRACT FROM AUTHOR]

Metabolic subtypes of glioblastoma (GBM) have different prognoses and responses to treatment. Deuterium metabolic imaging with 2H-labeled substrates is a potential approach to stratify patients into metabolic subtypes for targeted treatment. In this study, we used 2H magnetic resonance spectroscopy and magnetic resonance spectroscopic imaging (MRSI) measurements of [6,6'-2H2]glucose metabolism to identify metabolic subtypes and their responses to chemoradiotherapy in patient-derived GBM xenografts in vivo. The metabolism of patient-derived cells was first characterized in vitro by measuring the oxygen consumption rate, a marker of mitochondrial tricarboxylic acid cycle activity, as well as the extracellular acidification rate and 2H-labeled lactate production from [6,6'-2H2]glucose, which are markers of glycolytic activity. Two cell lines representative of a glycolytic subtype and two representative of a mitochondrial subtype were identified. 2H magnetic resonance spectroscopy and MRSI measurements showed similar concentrations of 2H-labeled glucose from [6,6'-2H2]glucose in all four tumor models when implanted orthotopically in mice. The glycolytic subtypes showed higher concentrations of 2H-labeled lactate than the mitochondrial subtypes and normal-appearing brain tissue, whereas the mitochondrial subtypes showed more glutamate/glutamine labeling, a surrogate for tricarboxylic acid cycle activity, than the glycolytic subtypes and normal-appearing brain tissue. The response of the tumors to chemoradiation could be detected within 24 hours of treatment completion, with the mitochondrial subtypes showing a decrease in both 2H-labeled glutamate/glutamine and lactate concentrations and glycolytic tumors showing a decrease in 2H-labeled lactate concentration. This technique has the potential to be used clinically for treatment selection and early detection of treatment response., Significance: Deuterium magnetic resonance spectroscopic imaging of glucose metabolism has the potential to differentiate between glycolytic and mitochondrial metabolic subtypes in glioblastoma and to evaluate early treatment responses, which could guide patient treatment., (©2024 The Authors; Published by the American Association for Cancer Research.)

Background: Branched-chain aminotransferase 1 (BCAT1) has been proposed to drive proliferation and invasion of isocitrate dehydrogenase ( IDH ) wild-type glioblastoma cells. However, the Cancer Genome Atlas (TCGA) dataset shows considerable variation in the expression of this enzyme in glioblastoma. The aim of this study was to determine the role of BCAT1 in driving the proliferation and invasion of glioblastoma cells and xenografts that have widely differing levels of BCAT1 expression and the mechanism responsible., Methods: The activity of BCAT1 was modulated in IDH wild-type patient-derived glioblastoma cell lines, and in orthotopically implanted tumors derived from these cells, to examine the effects of BCAT1 expression on tumor phenotype., Results: In cells with constitutively high BCAT1 expression and a glycolytic metabolic phenotype, inducible shRNA knockdown of the enzyme resulted in reduced proliferation and invasion by increasing the concentration of α-ketoglutarate, leading to reduced DNA methylation, HIF-1α destabilization, and reduced expression of the transcription factor Forkhead box protein M1 (FOXM1). Conversely, overexpression of the enzyme increased HIF-1α expression and promoted proliferation and invasion. However, in cells with an oxidative phenotype and very low constitutive expression of BCAT1 increased expression of the enzyme had no effect on invasion and reduced cell proliferation. This occurred despite an increase in HIF-1α levels and could be explained by decreased TCA cycle flux., Conclusions: There is a wide variation in BCAT1 expression in glioblastoma and its role in proliferation and invasion is dependent on tumor subtype., Competing Interests: The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

One of the hallmarks of cancer is metabolic reprogramming, including high levels of aerobic glycolysis (the Warburg effect). Pyruvate is a product of glucose metabolism, and 13 C-MR imaging of the metabolism of hyperpolarized (HP) [1- 13 C]pyruvate (HP 13 C-MRI) has been shown to be a potentially versatile tool for the clinical evaluation of tumor metabolism. Hyperpolarization of the 13 C nuclear spin can increase the sensitivity of detection by 4-5 orders of magnitude. Therefore, following intravenous injection, the location of hyperpolarized 13 C-labeled pyruvate in the body and its subsequent metabolism can be tracked using 13 C-MRI. Hyperpolarized [ 13 C]urea and [1,4- 13 C 2 ]fumarate are also likely to translate to the clinic in the near future as tools for imaging tissue perfusion and post-treatment tumor cell death, respectively. For clinical breast imaging, HP 13 C-MRI can be combined with 1 H-MRI to address the need for detailed anatomical imaging combined with improved functional tumor phenotyping and very early identification of patients not responding to standard and novel neoadjuvant treatments. If the technical complexity of the hyperpolarization process and the relatively high associated costs can be reduced, then hyperpolarized 13 C-MRI has the potential to become more widely available for large-scale clinical trials.

There is an unmet clinical need for imaging agents capable of detecting early evidence of tumor cell death, since the timing, extent, and distribution of cell death in tumors following treatment can give an indication of treatment outcome. We describe here 68 Ga-labeled C2Am, which is a phosphatidylserine-binding protein, for imaging tumor cell death in vivo using positron emission tomography (PET). A one-pot synthesis of 68 Ga-C2Am (20 min, 25 °C, >95% radiochemical purity) has been developed, using a NODAGA-maleimide chelator. The binding of 68 Ga-C2Am to apoptotic and necrotic tumor cells was assessed in vitro using human breast and colorectal cancer cell lines, and in vivo, using dynamic PET measurements in mice implanted subcutaneously with the colorectal tumor cells and treated with a TRAIL-R2 agonist. 68 Ga-C2Am showed predominantly renal clearance and low retention in the liver, spleen, small intestine, and bone and generated a tumor-to-muscle (T/m) ratio of 2.3 ± 0.4, at 2 h post probe administration and at 24 h following treatment. 68 Ga-C2Am has the potential to be used in the clinic as a PET tracer for assessing early treatment response in tumors.

Mutations in the TP53 gene are common in cancer with the R248Q missense mutation conferring an increased propensity to aggregate. Previous p53 aggregation studies showed that, at micromolar concentrations, protein unfolding to produce aggregation-prone species is the rate-determining step. Here we show that, at physiological concentrations, aggregation kinetics of insect cell-derived full-length wild-type p53 and p53R248Q are determined by a nucleation-growth model, rather than formation of aggregation-prone monomeric species. Self-seeding, but not cross-seeding, increases aggregation rate, confirming the aggregation process as rate determining. p53R248Q displays enhanced aggregation propensity due to decreased solubility and increased aggregation rate, forming greater numbers of larger amorphous aggregates that disrupt lipid bilayers and invokes an inflammatory response. These results suggest that p53 aggregation can occur under physiological conditions, a rate enhanced by R248Q mutation, and that aggregates formed can cause membrane damage and inflammation that may influence tumorigenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Early detection of tumor cell death in glioblastoma following treatment with chemoradiation has the potential to distinguish between true disease progression and pseudoprogression. Tumor cell death can be detected noninvasively in vivo by imaging the production of [2,3-2H2]malate from [2,3-2H2]fumarate using 2H magnetic resonance (MR) spectroscopic imaging. We show here that 2H MR spectroscopy and spectroscopic imaging measurements of [2,3-2H2]fumarate metabolism can detect tumor cell death in orthotopically implanted glioblastoma models within 48 hours following the completion of chemoradiation. Following the injection of [2,3-2H2]fumarate into tumor-bearing mice, production of [2,3-2H2]malate was measured in a human cell line-derived model and in radiosensitive and radioresistant patient-derived models of glioblastoma that were treated with temozolomide followed by targeted fractionated irradiation. The increase in the [2,3-2H2]malate/[2,3-2H2]fumarate signal ratio posttreatment, which correlated with histologic assessment of cell death, was a more sensitive indicator of treatment response than diffusion-weighted and contrast agent-enhanced 1H MRI measurements, which have been used clinically to detect responses of glioblastoma to chemoradiation. Overall, early detection of glioblastoma cell death using 2H MRI of malate production from fumarate could help improve the clinical evaluation of response to chemoradiation., Significance: 2H magnetic resonance imaging of labeled fumarate metabolism can detect early evidence of tumor cell death following chemoradiation, meeting a clinical need to reliably detect treatment response in glioblastoma., (©2022 The Authors; Published by the American Association for Cancer Research.)

Untargeted Nuclear Magnetic Resonance (NMR) metabolomics of polar extracts from the pancreata of a caerulin-induced mouse model of pancreatitis (Pt) and of a transgenic mouse model of pancreatic cancer (PCa) were used to find metabolic markers of Pt and to characterize the metabolic changes accompanying PCa progression. Using multivariate analysis a 10-metabolite metabolic signature specific to Pt tissue was found to distinguish the benign condition from both normal tissue and precancerous tissue (low grade pancreatic intraepithelial neoplasia, PanIN, lesions). The mice pancreata showed significant changes in the progression from normal tissue, through low-grade and high-grade PanIN lesions to pancreatic ductal adenocarcinoma (PDA). These included increased lactate production, amino acid changes consistent with enhanced anaplerosis, decreased concentrations of intermediates in membrane biosynthesis (phosphocholine and phosphoethanolamine) and decreased glycosylated uridine phosphates, reflecting activation of the hexosamine biosynthesis pathway and protein glycosylation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Carneiro, Pinto, Serrao, Barros, Brindle and Gil.)

Purpose To evaluate glioblastoma (GBM) metabolism by using hyperpolarized carbon 13 ( 13 C) MRI to monitor the exchange of the hyperpolarized 13 C label between injected [1- 13 C]pyruvate and tumor lactate and bicarbonate. Materials and Methods In this prospective study, seven treatment-naive patients (age [mean ± SD], 60 years ± 11; five men) with GBM were imaged at 3 T by using a dual-tuned 13 C-hydrogen 1 head coil. Hyperpolarized [1- 13 C]pyruvate was injected, and signal was acquired by using a dynamic MRI spiral sequence. Metabolism was assessed within the tumor, in the normal-appearing brain parenchyma (NABP), and in healthy volunteers by using paired or unpaired t tests and a Wilcoxon signed rank test. The Spearman ρ correlation coefficient was used to correlate metabolite labeling with lactate dehydrogenase A (LDH-A) expression and some immunohistochemical markers. The Benjamini-Hochberg procedure was used to correct for multiple comparisons. Results The bicarbonate-to-pyruvate (BP) ratio was lower in the tumor than in the contralateral NABP ( P < .01). The tumor lactate-to-pyruvate (LP) ratio was not different from that in the NABP ( P = .38). The LP and BP ratios in the NABP were higher than those observed previously in healthy volunteers ( P < .05). Tumor lactate and bicarbonate signal intensities were strongly correlated with the pyruvate signal intensity (ρ = 0.92, P < .001, and ρ = 0.66, P < .001, respectively), and the LP ratio was weakly correlated with LDH-A expression in biopsy samples (ρ = 0.43, P = .04). Conclusion Hyperpolarized 13 C MRI demonstrated variation in lactate labeling in GBM, both within and between tumors. In contrast, bicarbonate labeling was consistently lower in tumors than in the surrounding NABP. Keywords: Hyperpolarized 13 C MRI, Glioblastoma, Metabolism, Cancer, MRI, Neuro-oncology Supplemental material is available for this article. Published under a CC BY 4.0 license.

Hyperpolarised magnetic resonance imaging (HP  13 C-MRI) is an emerging clinical technique to detect [1- 13 C]lactate production in prostate cancer (PCa) following intravenous injection of hyperpolarised [1- 13 C]pyruvate. Here we differentiate clinically significant PCa from indolent disease in a low/intermediate-risk population by correlating [1- 13 C]lactate labelling on MRI with the percentage of Gleason pattern 4 (%GP4) disease. Using immunohistochemistry and spatial transcriptomics, we show that HP  13 C-MRI predominantly measures metabolism in the epithelial compartment of the tumour, rather than the stroma. MRI-derived tumour [1- 13 C]lactate labelling correlated with epithelial mRNA expression of the enzyme lactate dehydrogenase (LDHA and LDHB combined), and the ratio of lactate transporter expression between the epithelial and stromal compartments (epithelium-to-stroma MCT4). We observe similar changes in MCT4, LDHA, and LDHB between tumours with primary Gleason patterns 3 and 4 in an independent TCGA cohort. Therefore, HP  13 C-MRI can metabolically phenotype clinically significant disease based on underlying metabolic differences in the epithelial and stromal tumour compartments., (© 2022. The Author(s).)

Differentiating aggressive clear cell renal cell carcinoma (ccRCC) from indolent lesions is challenging using conventional imaging. This work prospectively compared the metabolic imaging phenotype of renal tumors using carbon-13 MRI following injection of hyperpolarized [1- 13 C]pyruvate (HP- 13 C-MRI) and validated these findings with histopathology. Nine patients with treatment-naïve renal tumors (6 ccRCCs, 1 liposarcoma, 1 pheochromocytoma, 1 oncocytoma) underwent pre-operative HP- 13 C-MRI and conventional proton ( 1 H) MRI. Multi-regional tissue samples were collected using patient-specific 3D-printed tumor molds for spatial registration between imaging and molecular analysis. The apparent exchange rate constant ( k PL ) between 13 C-pyruvate and 13 C-lactate was calculated. Immunohistochemistry for the pyruvate transporter (MCT1) from 44 multi-regional samples, as well as associations between MCT1 expression and outcome in the TCGA-KIRC dataset, were investigated. Increasing k PL in ccRCC was correlated with increasing overall tumor grade (ρ = 0.92, p = 0.009) and MCT1 expression (r = 0.89, p = 0.016), with similar results acquired from the multi-regional analysis. Conventional 1 H-MRI parameters did not discriminate tumor grades. The correlation between MCT1 and ccRCC grade was confirmed within a TCGA dataset ( p < 0.001), where MCT1 expression was a predictor of overall and disease-free survival. In conclusion, metabolic imaging using HP- 13 C-MRI differentiates tumor aggressiveness in ccRCC and correlates with the expression of MCT1, a predictor of survival. HP- 13 C-MRI may non-invasively characterize metabolic phenotypes within renal cancer.

Radioligand theranostics (RT) in oncology use cancer-type specific biomarkers and molecular imaging (MI), including positron emission tomography (PET), single-photon emission computed tomography (SPECT) and planar scintigraphy, for patient diagnosis, therapy, and personalized management. While the definition of theranostics was initially restricted to a single compound allowing visualization and therapy simultaneously, the concept has been widened with the development of theranostic pairs and the combination of nuclear medicine with different types of cancer therapies. Here, we review the clinical applications of different theranostic radiopharmaceuticals in managing different tumor types (differentiated thyroid, neuroendocrine prostate, and breast cancer) that support the combination of innovative oncological therapies such as gene and cell-based therapies with RT.

Hyperpolarized 13 C-MRI is an emerging tool for probing tissue metabolism by measuring 13 C-label exchange between intravenously injected hyperpolarized [1- 13 C]pyruvate and endogenous tissue lactate. Here, we demonstrate that hyperpolarized 13 C-MRI can be used to detect early response to neoadjuvant therapy in breast cancer. Seven patients underwent multiparametric 1 H-MRI and hyperpolarized 13 C-MRI before and 7-11 days after commencing treatment. An increase in the lactate-to-pyruvate ratio of approximately 20% identified three patients who, following 5-6 cycles of treatment, showed pathological complete response. This ratio correlated with gene expression of the pyruvate transporter MCT1 and lactate dehydrogenase A ( LDHA ), the enzyme catalyzing label exchange between pyruvate and lactate. Analysis of approximately 2,000 breast tumors showed that overexpression of LDHA and the hypoxia marker CAIX was associated with reduced relapse-free and overall survival. Hyperpolarized 13 C-MRI represents a promising method for monitoring very early treatment response in breast cancer and has demonstrated prognostic potential. SIGNIFICANCE: Hyperpolarized carbon-13 MRI allows response assessment in patients with breast cancer after 7-11 days of neoadjuvant chemotherapy and outperformed state-of-the-art and research quantitative proton MRI techniques., (©2021 The Authors; Published by the American Association for Cancer Research.)

Background: Immune checkpoint inhibitors are now standard of care treatment for many cancers. Treatment failure in metastatic melanoma is often due to tumor heterogeneity, which is not easily captured by conventional CT or tumor biopsy. The aim of this prospective study was to investigate early microstructural and functional changes within melanoma metastases following immune checkpoint blockade using multiparametric MRI., Methods: Fifteen treatment-naïve metastatic melanoma patients (total 27 measurable target lesions) were imaged at baseline and following 3 and 12 weeks of treatment on immune checkpoint inhibitors using: T 2 -weighted imaging, diffusion kurtosis imaging, and dynamic contrast-enhanced MRI. Treatment timepoint changes in tumor cellularity, vascularity, and heterogeneity within individual metastases were evaluated and correlated to the clinical outcome in each patient based on Response Evaluation Criteria in Solid Tumors V.1.1 at 1 year., Results: Differential tumor growth kinetics in response to immune checkpoint blockade were measured in individual metastases within the same patient, demonstrating significant intertumoral heterogeneity in some patients. Early detection of tumor cell death or cell loss measured by a significant increase in the apparent diffusivity (D app ) (p<0.05) was observed in both responding and pseudoprogressive lesions after 3 weeks of treatment. Tumor heterogeneity, as measured by apparent diffusional kurtosis (K app ), was consistently higher in the pseudoprogressive and true progressive lesions, compared with the responding lesions throughout the first 12 weeks of treatment. These preceded tumor regression and significant tumor vascularity changes (K trans , v e , and v p ) detected after 12 weeks of immunotherapy (p<0.05)., Conclusions: Multiparametric MRI demonstrated potential for early detection of successful response to immune checkpoint inhibitors in metastatic melanoma., Competing Interests: Competing interests: No conflict of interest to declare with respect to the content of this work. AL, MS, LB, and J-ML are employees of AstraZeneca UK., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

13 C nuclear spin hyperpolarization can increase the sensitivity of detection in an MRI experiment by more than 10,000-fold. 13 C magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized 13 C label exchange between injected [1- 13 C]pyruvate and the endogenous tumor lactate pool can be used clinically to assess tumor grade and response to treatment. We describe here an experimental protocol for using this technique in patient-derived and established cell line xenograft models of breast cancer in the mouse. For complete details on the use and execution of this protocol, please refer to Ros et al. (2020)., Competing Interests: The hyperpolarizer is on loan from GE Healthcare (GEH) and is the subject of a research agreement between the University of Cambridge, Cancer Research UK, and GEH. K.M.B. holds patents with GEH on some aspects of dissolution dynamic nuclear polarization (DNP) technology. C.C. reports receiving speakers' bureau honoraria from Illumina, is a member of the AstraZeneca (AZ) External Science Panel, and reports receiving research grants from Roche, Genentech, AZ, and Servier that are administered by the University of Cambridge., (© 2021 The Authors.)

2 H magnetic resonance spectroscopic imaging has been shown recently to be a viable technique for metabolic imaging in the clinic. We show here that 2 H MR spectroscopy and spectroscopic imaging measurements of [2,3- 2 H 2 ]malate production from [2,3- 2 H 2 ]fumarate can be used to detect tumor cell death in vivo via the production of labeled malate. Production of [2,3- 2 H 2 ]malate, following injection of [2,3- 2 H 2 ]fumarate (1 g/kg) into tumor-bearing mice, was measured in a murine lymphoma (EL4) treated with etoposide, and in human breast (MDA-MB-231) and colorectal (Colo205) xenografts treated with a TRAILR2 agonist, using surface-coil localized 2 H MR spectroscopy at 7 T. Malate production was also imaged in EL4 tumors using a fast 2 H chemical shift imaging sequence. The malate/fumarate ratio increased from 0.016 ± 0.02 to 0.16 ± 0.14 in EL4 tumors 48 h after drug treatment ( P = 0.0024, n = 3), and from 0.019 ± 0.03 to 0.25 ± 0.23 in MDA-MB-231 tumors ( P = 0.0001, n = 5) and from 0.016 ± 0.04 to 0.28 ± 0.26 in Colo205 tumors ( P = 0.0002, n = 5) 24 h after drug treatment. These increases were correlated with increased levels of cell death measured in excised tumor sections obtained immediately after imaging. 2 H MR measurements of [2,3- 2 H 2 ]malate production from [2,3- 2 H 2 ]fumarate provide a potentially less expensive and more sensitive method for detecting cell death in vivo than 13 C MR measurements of hyperpolarized [1,4- 13 C 2 ]fumarate metabolism, which have been used previously for this purpose., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Ductal carcinoma in situ (DCIS) is a non-invasive form of early breast cancer, with a poorly understood natural history of invasive transformation. Necrosis is a well-recognized adverse prognostic feature of DCIS, and non-invasive detection of its presence and spatial extent could provide information not obtainable by biopsy. We describe here imaging of the distribution and extent of comedo-type necrosis in a model of human DCIS using C2Am, an imaging agent that binds to the phosphatidylserine exposed by necrotic cells., Methods: We used an established xenograft model of human DCIS that mimics the histopathological features of the disease. Planar near-infrared and optoacoustic imaging, using fluorescently labeled C2Am, were used to image non-invasively the presence and extent of lesion necrosis., Results: C2Am showed specific and sensitive binding to necrotic areas in DCIS tissue, detectable both in vivo and ex vivo. The imaging signal generated in vivo using near-infrared (NIR) fluorescence imaging was up to 6-fold higher in DCIS lesions than in surrounding fat pad or skin tissue. There was a correlation between the C2Am NIR fluorescence (Pearson R = 0.783, P = 0.0125) and optoacoustic signals (R > 0.875, P < 0.022) in the DCIS lesions in vivo and the corresponding levels of cell death detected histologically., Conclusions: C2Am is a targeted multi-modal imaging agent that could complement current anatomical imaging methods for detecting DCIS. Imaging the presence and spatial extent of necrosis may give better prognostic information than that obtained by biopsy alone.

Introduction: Trialing novel cancer therapies in the clinic would benefit from imaging agents that can detect early evidence of treatment response. The timing, extent and distribution of cell death in tumors following treatment can give an indication of outcome. We describe here an 18 F-labeled derivative of a phosphatidylserine-binding protein, the C2A domain of Synaptotagmin-I (C2Am), for imaging tumor cell death in vivo using PET., Methods: A one-pot, two-step automated synthesis of N-(5-[ 18 F]fluoropentyl)maleimide (60 min synthesis time, > 98% radiochemical purity) has been developed, which was used to label the single cysteine residue in C2Am within 30 min at room temperature. Binding of 18 F-C2Am to apoptotic and necrotic tumor cells was assessed in vitro, and also in vivo, by dynamic PET and biodistribution measurements in mice bearing human tumor xenografts treated with a TRAILR2 agonist or with conventional chemotherapy. C2Am detection of tumor cell death was validated by correlation of probe binding with histological markers of cell death in tumor sections obtained immediately after imaging., Results: 18 F-C2Am showed a favorable biodistribution profile, with predominantly renal clearance and minimal retention in spleen, liver, small intestine, bone and kidney, at 2 h following probe administration. 18 F-C2Am generated tumor-to-muscle (T/m) ratios of 6.1 ± 2.1 and 10.7 ± 2.4 within 2 h of probe administration in colorectal and breast tumor models, respectively, following treatment with the TRAILR2 agonist. The levels of cell death (CC3 positivity) following treatment were 12.9-58.8% and 11.3-79.7% in the breast and colorectal xenografts, respectively. Overall, a 20% increase in CC3 positivity generated a one unit increase in the post/pre-treatment tumor contrast. Significant correlations were found between tracer uptake post-treatment, at 2 h post-probe administration, and histological markers of cell death (CC3: Pearson R = 0.733, P = 0.0005; TUNEL: Pearson R = 0.532, P = 0.023)., Conclusion: The rapid clearance of 18 F-C2Am from the blood pool and low kidney retention allowed the spatial distribution of cell death in a tumor to be imaged during the course of therapy, providing a rapid assessment of tumor treatment response. 18 F-C2Am has the potential to be used in the clinic to assess early treatment response in tumors.

Magnetic resonance imaging of the pancreas is increasingly used as an important diagnostic modality for characterisation of pancreatic lesions. Pancreatic MRI protocols are mostly qualitative due to time constraints and motion sensitivity. MR Fingerprinting is an innovative acquisition technique that provides qualitative data and quantitative parameter maps from a single free-breathing acquisition with the potential to reduce exam times. This work investigates the feasibility of MRF parameter mapping for pancreatic imaging in the presence of free-breathing exam. Sixteen healthy participants were prospectively imaged using MRF framework. Regions-of-interest were drawn in multiple solid organs including the pancreas and T 1 and T 2  values determined. MRF T 1 and T 2 mapping was performed successfully in all participants (acquisition time:2.4-3.6 min). Mean pancreatic T 1 values were 37-43% lower than those of the muscle, spleen, and kidney at both 1.5 and 3.0 T. For these organs, the mean pancreatic T 2 values were nearly 40% at 1.5 T and < 12% at 3.0 T. The feasibility of MRF at 1.5 T and 3 T was demonstrated in the pancreas. By enabling fast and free-breathing quantitation, MRF has the potential to add value during the clinical characterisation and grading of pathological conditions, such as pancreatitis or cancer.

PIK3CA, encoding the PI3Kα isoform, is the most frequently mutated oncogene in estrogen receptor (ER)-positive breast cancer. Isoform-selective PI3K inhibitors are used clinically but intrinsic and acquired resistance limits their utility. Improved selection of patients that will benefit from these drugs requires predictive biomarkers. We show here that persistent FOXM1 expression following drug treatment is a biomarker of resistance to PI3Kα inhibition in ER + breast cancer. FOXM1 drives expression of lactate dehydrogenase (LDH) but not hexokinase 2 (HK-II). The downstream metabolic changes can therefore be detected using MRI of LDH-catalyzed hyperpolarized 13 C label exchange between pyruvate and lactate but not by positron emission tomography measurements of HK-II-mediated trapping of the glucose analog 2-deoxy-2-[ 18 F]fluorodeoxyglucose. Rapid assessment of treatment response in breast cancer using this imaging method could help identify patients that benefit from PI3Kα inhibition and design drug combinations to counteract the emergence of resistance., Competing Interests: Declaration of Interests The hyperpolarizer is on loan from GE Healthcare (GEH) and is the subject of a research agreement between the University of Cambridge, Cancer Research UK, and GEH. K.M.B holds patents with GEH on some aspects of deoxyribonucleoprotein (DNP) technology and is a consultant for Sotio a.s. C.C. reports receiving speakers' bureau honoraria from Illumina, is a member of the AstraZeneca (AZ) External Science Panel, and has research grants from Roche, Genentech, AZ, and Servier that are administered by the University of Cambridge. R.D.B reports receiving speakers’ bureau honoraria from Novartis and Roche/Genentech; commercial research grants from Genentech, AZ, and Boehringer-Ingelheim; and commercial research support from AZ. M.O. reports receiving speakers' bureau honoraria from Roche and is a consultant/advisory board member for Roche, GlaxoSmithKline, and Puma Biotechnology. S.C.L. is an advisory board member for AZ, Cergentis, IBM, Pfizer, and Roche and received institutional research support from Agendia, AZ, Eurocept Pharmaceuticals, Genentech, Novartis, Pfizer, Roche, Tesaro, and Immunomedics. In addition, S.C.L. received institutional non-financial support from Genentech, Novartis, Roche, Tesaro, and Immunomedics and other institutional support from AZ, Pfizer, Cergentis, IBM, and Bayer outside of this study. P.R. has received consultation fees from Novartis, AZ, and Foundation Medicine and institutional research funds from Grail Inc, Novartis, and Illumina. M.S. has received research funds from Puma Biotechnology, Daiichi-Sankio, Immunomedics, Targimmune, and Menarini Ricerche; is a cofounder of Medendi Medical Travel; and serves on the advisory board of Menarini Ricerche., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: To compare hyperpolarized carbon 13 ( 13 C) MRI with dynamic contrast material-enhanced (DCE) MRI in the detection of early treatment response in breast cancer., Materials and Methods: In this institutional review board-approved prospective study, a woman with triple-negative breast cancer (age, 49 years) underwent 13 C MRI after injection of hyperpolarized [1-carbon 13 { 13 C}]-pyruvate and DCE MRI at 3 T at baseline and after one cycle of neoadjuvant therapy. The 13 C-labeled lactate-to-pyruvate ratio derived from hyperpolarized 13 C MRI and the pharmacokinetic parameters transfer constant ( K trans ) and washout parameter ( k ep ) derived from DCE MRI were compared before and after treatment., Results: Exchange of the 13 C label between injected hyperpolarized [1- 13 C]-pyruvate and the endogenous lactate pool was observed, catalyzed by the enzyme lactate dehydrogenase. After one cycle of neoadjuvant chemotherapy, a 34% reduction in the 13 C-labeled lactate-to-pyruvate ratio resulted in correct identification of the patient as a responder to therapy, which was subsequently confirmed via a complete pathologic response. However, DCE MRI showed an increase in mean K trans (132%) and mean k ep (31%), which could be incorrectly interpreted as a poor response to treatment., Conclusion: Hyperpolarized 13 C MRI enabled successful identification of breast cancer response after one cycle of neoadjuvant chemotherapy and may improve response prediction when used in conjunction with multiparametric proton MRI.Published under a CC BY 4.0 license., (2020 by the Radiological Society of North America, Inc.)

Rationale: The recent development of hyperpolarized 13 C magnetic resonance spectroscopy has made it possible to measure cellular metabolism in vivo, in real time., Objective: By comparing participants with and without type 2 diabetes mellitus (T2DM), we report the first case-control study to use this technique to record changes in cardiac metabolism in the healthy and diseased human heart., Methods and Results: Thirteen people with T2DM (glycated hemoglobin, 6.9±1.0%) and 12 age-matched healthy controls underwent assessment of cardiac systolic and diastolic function, myocardial energetics ( 31 P-magnetic resonance spectroscopy), and lipid content ( 1 H-magnetic resonance spectroscopy) in the fasted state. In a subset (5 T2DM, 5 control), hyperpolarized [1- 13 C]pyruvate magnetic resonance spectra were also acquired and in 5 of these participants (3 T2DM, 2 controls), this was successfully repeated 45 minutes after a 75 g oral glucose challenge. Downstream metabolism of [1- 13 C]pyruvate via PDH (pyruvate dehydrogenase, [ 13 C]bicarbonate), lactate dehydrogenase ([1- 13 C]lactate), and alanine transaminase ([1- 13 C]alanine) was assessed. Metabolic flux through cardiac PDH was significantly reduced in the people with T2DM (Fasted: 0.0084±0.0067 [Control] versus 0.0016±0.0014 [T2DM], Fed: 0.0184±0.0109 versus 0.0053±0.0041; P =0.013). In addition, a significant increase in metabolic flux through PDH was observed after the oral glucose challenge ( P <0.001). As is characteristic of diabetes mellitus, impaired myocardial energetics, myocardial lipid content, and diastolic function were also demonstrated in the wider study cohort., Conclusions: This work represents the first demonstration of the ability of hyperpolarized 13 C magnetic resonance spectroscopy to noninvasively assess physiological and pathological changes in cardiac metabolism in the human heart. In doing so, we highlight the potential of the technique to detect and quantify metabolic alterations in the setting of cardiovascular disease.

Background Tumor cells frequently show high rates of aerobic glycolysis, which provides the glycolytic intermediates needed for the increased biosynthetic demands of rapid cell growth and proliferation. Existing clinical methods (fluorodeoxyglucose PET and carbon 13 MRI and spectroscopy) do not allow quantitative images of glycolytic flux. Purpose To evaluate the use of deuterium (hydrogen 2 [ 2 H]) MR spectroscopic imaging for quantitative mapping of tumor glycolytic flux and to assess response to chemotherapy. Materials and Methods A fast three-dimensional 2 H MR spectroscopic imaging pulse sequence, with a time resolution of 10 minutes, was used to image glycolytic flux in a murine tumor model after bolus injection of D-[6,6'- 2 H 2 ]glucose before and 48 hours after treatment with a chemotherapeutic agent. Tumor lactate labeling, expressed as the lactate-to-water and lactate-to-glucose signal ratios, was also assessed in localized 2 H MR spectra. Statistical significance was tested with a one-sided paired t test. Results 2 H MR spectroscopic imaging showed heterogeneity in glycolytic flux across the tumor and an early decrease in flux following treatment with a chemotherapeutic drug. Spectroscopy measurements on five animals showed a decrease in the lactate-to-water signal ratio, from 0.33 ± 0.10 to 0.089 ± 0.039 ( P = .005), and in the lactate-to-glucose ratio, from 0.27 ± 0.12 to 0.12 ± 0.06 ( P = .04), following drug treatment. Conclusion Rapidly acquired deuterium (hydrogen 2) MR spectroscopic images can provide quantitative and spatially resolved measurements of glycolytic flux in tumors that can be used to assess treatment response. Published under a CC BY 4.0 license. Online supplemental material is available for this article. See also the editorial by Ouwerkerk in this issue.

Our purpose is to investigate the feasibility of imaging tumor metabolism in breast cancer patients using 13 C magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized 13 C label exchange between injected [1- 13 C]pyruvate and the endogenous tumor lactate pool. Treatment-naïve breast cancer patients were recruited: four triple-negative grade 3 cancers; two invasive ductal carcinomas that were estrogen and progesterone receptor-positive (ER/PR+) and HER2/neu-negative (HER2-), one grade 2 and one grade 3; and one grade 2 ER/PR+ HER2- invasive lobular carcinoma (ILC). Dynamic 13 C MRSI was performed following injection of hyperpolarized [1- 13 C]pyruvate. Expression of lactate dehydrogenase A (LDHA), which catalyzes 13 C label exchange between pyruvate and lactate, hypoxia-inducible factor-1 (HIF1α), and the monocarboxylate transporters MCT1 and MCT4 were quantified using immunohistochemistry and RNA sequencing. We have demonstrated the feasibility and safety of hyperpolarized 13 C MRI in early breast cancer. Both intertumoral and intratumoral heterogeneity of the hyperpolarized pyruvate and lactate signals were observed. The lactate-to-pyruvate signal ratio (LAC/PYR) ranged from 0.021 to 0.473 across the tumor subtypes (mean ± SD: 0.145 ± 0.164), and a lactate signal was observed in all of the grade 3 tumors. The LAC/PYR was significantly correlated with tumor volume ( R = 0.903, P = 0.005) and MCT 1 ( R = 0.85, P = 0.032) and HIF1α expression ( R = 0.83, P = 0.043). Imaging of hyperpolarized [1- 13 C]pyruvate metabolism in breast cancer is feasible and demonstrated significant intertumoral and intratumoral metabolic heterogeneity, where lactate labeling correlated with MCT1 expression and hypoxia., Competing Interests: Competing interest statement: A research agreement is in place between GE Healthcare and K.M.B. and F.A.G., (Copyright © 2020 the Author(s). Published by PNAS.)

Metabolic imaging has been widely used to measure the early responses of tumors to treatment. Here, we assess the abilities of PET measurement of [ 18 F]FDG uptake and MRI measurement of hyperpolarized [1- 13 C]pyruvate metabolism to detect early changes in glycolysis following treatment-induced cell death in human colorectal (Colo205) and breast adenocarcinoma (MDA-MB-231) xenografts in mice. A TRAIL agonist that binds to human but not mouse cells induced tumor-selective cell death. Tumor glycolysis was assessed by injecting [1,6- 13 C 2 ]glucose and measuring 13 C-labeled metabolites in tumor extracts. Injection of hyperpolarized [1- 13 C]pyruvate induced rapid reduction in lactate labeling. This decrease, which correlated with an increase in histologic markers of cell death and preceded decrease in tumor volume, reflected reduced flux from glucose to lactate and decreased lactate concentration. However, [ 18 F]FDG uptake and phosphorylation were maintained following treatment, which has been attributed previously to increased [ 18 F]FDG uptake by infiltrating immune cells. Quantification of [ 18 F]FDG uptake in flow-sorted tumor and immune cells from disaggregated tumors identified CD11b + /CD45 + macrophages as the most [ 18 F]FDG-avid cell type present, yet they represented <5% of the cells present in the tumors and could not explain the failure of [ 18 F]FDG-PET to detect treatment response. MRI measurement of hyperpolarized [1- 13 C]pyruvate metabolism is therefore a more sensitive marker of the early decreases in glycolytic flux that occur following cell death than PET measurements of [ 18 F]FDG uptake. SIGNIFICANCE: These findings demonstrate superior sensitivity of MRI measurement of hyperpolarized [1- 13 C]pyruvate metabolism versus PET measurement of 18 F-FDG uptake for detecting early changes in glycolysis following treatment-induced tumor cell death., (©2019 American Association for Cancer Research.)

Due to the implication of altered metabolism in a large spectrum of tissue function and disease, assessment of metabolic processes becomes essential in managing health. In this regard, imaging can play a critical role in allowing observation of biochemical and physiological processes. Nuclear imaging methods, in particular positron emission tomography, have been widely employed for imaging metabolism but are mainly limited by the use of ionizing radiation and the sensing of only one parameter at each scanning session. Observations in healthy individuals or longitudinal studies of disease could markedly benefit from non-ionizing, multi-parameter imaging methods. We therefore focus this review on progress with the non-ionizing radiation methods of MRI, hyperpolarized magnetic resonance and magnetic resonance spectroscopy, chemical exchange saturation transfer, and emerging optoacoustic (photoacoustic) imaging. We also briefly discuss the role of nuclear and optical imaging methods for research and clinical protocols., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The factors responsible for the low detection rate of cell-free tumor DNA (ctDNA) in the plasma of patients with glioblastoma (GBM) are currently unknown. In this study, we measured circulating nucleic acids in patient-derived orthotopically implanted xenograft (PDOX) models of GBM ( n = 64) and show that tumor size and cell proliferation, but not the integrity of the blood-brain barrier or cell death, affect the release of ctDNA in treatment-naïve GBM PDOX. Analysis of fragment length profiles by shallow genome-wide sequencing (<0.2× coverage) of host (rat) and tumor (human) circulating DNA identified a peak at 145 bp in the human DNA fragments, indicating a difference in the origin or processing of the ctDNA. The concentration of ctDNA correlated with cell death only after treatment with temozolomide and radiotherapy. Digital PCR detection of plasma tumor mitochondrial DNA (tmtDNA), an alternative to detection of nuclear ctDNA, improved plasma DNA detection rate (82% vs. 24%) and allowed detection in cerebrospinal fluid and urine. Mitochondrial mutations are prevalent across all cancers and can be detected with high sensitivity, at low cost, and without prior knowledge of tumor mutations via capture-panel sequencing. Coupled with the observation that mitochondrial copy number increases in glioma, these data suggest analyzing tmtDNA as a more sensitive method to detect and monitor tumor burden in cancer, specifically in GBM, where current methods have largely failed. SIGNIFICANCE: These findings show that detection of tumor mitochondrial DNA is more sensitive than circulating tumor DNA analysis to detect and monitor tumor burden in patient-derived orthotopic xenografts of glioblastoma., (©2018 American Association for Cancer Research.)

Methylglyoxal is a faulty metabolite. It is a ubiquitous by-product of glucose and amino acid metabolism that spontaneously reacts with proximal amino groups in proteins and nucleic acids, leading to impairment of their function. The glyoxalase pathway evolved early in phylogeny to bring about rapid catabolism of methylglyoxal, and an understanding of the role of methylglyoxal and the glyoxalases in many diseases is beginning to emerge. Metabolic processing of methylglyoxal is very rapid in vivo and thus notoriously difficult to detect and quantify. Here we show that 13 C nuclei in labeled methylglyoxal can be hyperpolarized using dynamic nuclear polarization, providing 13 C nuclear magnetic resonance signal enhancements in the solution state close to 5,000-fold. We demonstrate the applications of this probe of metabolism for kinetic characterization of the glyoxalase system in isolated cells as well as mouse brain, liver and lymphoma in vivo., Competing Interests: The authors declare no competing interests.

Glioma is difficult to detect or characterize using current liquid biopsy approaches. Detection of cell-free tumor DNA (cftDNA) in cerebrospinal fluid (CSF) has been proposed as an alternative to detection in plasma. We used shallow whole-genome sequencing (sWGS, at a coverage of < 0.4×) of cell-free DNA from the CSF of 13 patients with primary glioma to determine somatic copy number alterations and DNA fragmentation patterns. This allowed us to determine the presence of cftDNA in CSF without any prior knowledge of point mutations present in the tumor. We also showed that the fragmentation pattern of cell-free DNA in CSF is different from that in plasma. This low-cost screening method provides information on the tumor genome and can be used to target those patients with high levels of cftDNA for further larger-scale sequencing, such as by whole-exome and whole-genome sequencing., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

13 C MRI of hyperpolarized [1- 13 C]pyruvate metabolism has been used in oncology to detect disease, investigate disease progression, and monitor response to treatment with a view to guiding treatment in individual patients. This technique has translated to the clinic with initial studies in prostate cancer. Here, we use the technique to investigate its potential uses in patients with glioblastoma (GB). We assessed the metabolism of hyperpolarized [1- 13 C]pyruvate in an orthotopically implanted cell line model (U87) of GB and in patient-derived tumors, where these were produced by orthotopic implantation of cells derived from different patients. Lactate labeling was higher in the U87 tumor when compared with patient-derived tumors, which displayed intertumoral heterogeneity, reflecting the intra- and intertumoral heterogeneity in the patients' tumors from which they were derived. Labeling in some patient-derived tumors could be observed before their appearance in morphologic images, whereas in other tumors it was not significantly greater than the surrounding brain. Increased lactate labeling in tumors correlated with c-Myc-driven expression of hexokinase 2, lactate dehydrogenase A, and the monocarboxylate transporters and was accompanied by increased radioresistance. Because c-Myc expression correlates with glioma grade, this study demonstrates that imaging with hyperpolarized [1- 13 C]pyruvate could be used clinically with patients with GB to determine disease prognosis, to detect early responses to drugs that modulate c-Myc expression, and to select tumors, and regions of tumors for increased radiotherapy dose. Significance: Metabolic imaging with hyperpolarized [1- 13 C]pyruvate detects low levels of c-Myc-driven glycolysis in patient-derived glioblastoma models, which, when translated to the clinic, could be used to detect occult disease, determine disease prognosis, and target radiotherapy. Cancer Res; 78(18); 5408-18. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The extent of surgical resection is significantly correlated with outcome in glioma; however, current intraoperative navigational tools are useful only in a subset of patients. We show here that a new optical intraoperative technique, Cerenkov luminescence imaging (CLI) following intravenous injection of O‑(2-[ 18 F]fluoroethyl)-L-tyrosine (FET), can be used to accurately delineate glioma margins, performing better than the current standard of fluorescence imaging with 5-aminolevulinic acid (5-ALA). Methods: Rats implanted orthotopically with U87, F98 and C6 glioblastoma cells were injected with FET and 5-aminolevulinic acid (5-ALA). Positive and negative tumor regions on histopathology were compared with CL and fluorescence images. The capability of FET CLI and 5-ALA fluorescence imaging to detect tumor was assessed using receptor operator characteristic curves and optimal thresholds (CLI OptROC and 5-ALA OptROC ) separating tumor from healthy brain tissue were determined. These thresholds were used to guide prospective tumor resections, where the presence of tumor cells in the resected material and in the remaining brain were assessed by Ki-67 staining. Results: FET CLI signal was correlated with signal in preoperative PET images (y = 1.06x - 0.01; p < 0.0001) and with expression of the amino acid transporter SLC7A5 (LAT1). FET CLI (AUC = 97%) discriminated between glioblastoma and normal brain in human and rat orthografts more accurately than 5-ALA fluorescence (AUC = 91%), with a sensitivity >92% and specificity >91%, and resulted in a more complete tumor resection. Conclusion: FET CLI can be used to accurately delineate glioblastoma tumor margins, performing better than the current standard of fluorescence imaging following 5-ALA administration, and is therefore a promising technique for clinical translation., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Correction: Unfortunately, the original version of Figs. 4, 5 and 6b in the article [1] contained errors in the n numbers as indicated on the columns. Please note that column heights and error bars in the original figures and data in the ESM tables are correct and statistical tests are valid. These corrections do not affect any results or conclusions in this article.

Purpose: The development of new treatments and their deployment in the clinic may be assisted by imaging methods that allow an early assessment of treatment response in individual patients. The C2A domain of Synaptotagmin-I (C2Am), which binds to the phosphatidylserine (PS) exposed by apoptotic and necrotic cells, has been developed as an imaging probe for detecting cell death. Multispectral optoacoustic tomography (MSOT) is a real-time and clinically applicable imaging modality that was used here with a near infrared (NIR) fluorophore-labeled C2Am to image tumor cell death in mice treated with a TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) agonist and with 5-fluorouracil (5-FU). Experimental Design: C2Am was labeled with a NIR fluorophore and injected intravenously into mice bearing human colorectal TRAIL-sensitive Colo205 and TRAIL-resistant HT-29 xenografts that had been treated with a potent agonist of TRAILR2 and in Colo205 tumors treated with 5-FU. Results: Three-dimensional (3D) MSOT images of probe distribution showed development of tumor contrast within 3 hours of probe administration and a signal-to-background ratio in regions containing dead cells of >10 after 24 hours. A site-directed mutant of C2Am that is inactive in PS binding showed negligible binding. Tumor retention of the active probe was strongly correlated ( R 2 = 0.97, P value < 0.01) with a marker of apoptotic cell death measured in histologic sections obtained post mortem. Conclusions: The rapid development of relatively high levels of contrast suggests that NIR fluorophore-labeled C2Am could be a useful optoacoustic imaging probe for detecting early therapy-induced tumor cell death in the clinic. Clin Cancer Res; 23(22); 6893-903. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 (PFKFB4) controls metabolic flux through allosteric regulation of glycolysis. Here we show that p53 regulates the expression of PFKFB4 and that p53-deficient cancer cells are highly dependent on the function of this enzyme. We found that p53 downregulates PFKFB4 expression by binding to its promoter and mediating transcriptional repression via histone deacetylases. Depletion of PFKFB4 from p53-deficient cancer cells increased levels of the allosteric regulator fructose-2,6-bisphosphate, leading to increased glycolytic activity but decreased routing of metabolites through the oxidative arm of the pentose-phosphate pathway. PFKFB4 was also required to support the synthesis and regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) in p53-deficient cancer cells. Moreover, depletion of PFKFB4-attenuated cellular biosynthetic activity and resulted in the accumulation of reactive oxygen species and cell death in the absence of p53. Finally, silencing of PFKFB4-induced apoptosis in p53-deficient cancer cells in vivo and interfered with tumour growth. These results demonstrate that PFKFB4 is essential to support anabolic metabolism in p53-deficient cancer cells and suggest that inhibition of PFKFB4 could be an effective strategy for cancer treatment.

Cell death is an important target for imaging the early response of tumors to treatment. We describe here the validation of a phosphatidylserine-binding agent for detecting tumor cell death in vivo based on the C2A domain of synaptotagmin-I. Methods: The capability of near-infrared fluorophore-labeled and 99m Tc- and 111 In-labeled derivatives of C2Am for imaging tumor cell death, using planar near-infrared fluorescence imaging and SPECT, respectively, was evaluated in implanted and genetically engineered mouse models of lymphoma and in a human colorectal xenograft. Results: The fluorophore-labeled C2Am derivative showed predominantly renal clearance and high specificity and sensitivity for detecting low levels of tumor cell death (2%-5%). There was a significant correlation ( R > 0.9, P < 0.05) between fluorescently labeled C2Am binding and histologic markers of cell death, including cleaved caspase-3, whereas there was no such correlation with a site-directed mutant of C2Am (iC2Am) that does not bind phosphatidylserine. 99m Tc-C2Am and 111 In-C2Am also showed favorable biodistribution profiles, with predominantly renal clearance and low nonspecific retention in the liver and spleen at 24 h after probe administration. 99m Tc-C2Am and 111 In-C2Am generated tumor-to-muscle ratios in drug-treated tumors of 4.3× and 2.2×, respectively, at 2 h and 7.3× and 4.1×, respectively, at 24 h after administration. Conclusion: Given the favorable biodistribution profile of 99m Tc- and 111 In-labeled C2Am, and their ability to produce rapid and cell death-specific image contrast, these agents have potential for clinical translation., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

Purpose: To develop an image analysis technique that distinguishes pseudoprogression from true progression by analyzing tumour heterogeneity in T2-weighted images using topological descriptors of image heterogeneity called Minkowski functionals (MFs)., Methods: Using a retrospective patient cohort (n = 50), and blinded to treatment response outcome, unsupervised feature estimation was performed to investigate MFs for the presence of outliers, potential confounders, and sensitivity to treatment response. The progression and pseudoprogression groups were then unblinded and supervised feature selection was performed using MFs, size and signal intensity features. A support vector machine model was obtained and evaluated using a prospective test cohort., Results: The model gave a classification accuracy, using a combination of MFs and size features, of more than 85% in both retrospective and prospective datasets. A different feature selection method (Random Forest) and classifier (Lasso) gave the same results. Although not apparent to the reporting radiologist, the T2-weighted hyperintensity phenotype of those patients with progression was heterogeneous, large and frond-like when compared to those with pseudoprogression., Conclusion: Analysis of heterogeneity, in T2-weighted MR images, which are acquired routinely in the clinic, has the potential to detect an earlier treatment response allowing an early change in treatment strategy. Prospective validation of this technique in larger datasets is required.

As clinical oncology evolves with new treatment options becoming available, there is an increasing demand on anatomic imaging for the assessment of patients at different stages. Imaging with hyperpolarized 13 C-labelled cell substrates has the potential to become a powerful tool in many steps of clinical evaluation, offering a new metabolic metric and therefore a more personalised approach to treatment response. This articles explores the metabolic basis and potential for translation of hyperpolarised pyruvate as a dynamic nuclear polarisation probe in clinical oncology., Competing Interests: KB's lab has a research agreement with GE Healthcare (GEH) and holds patents on DNP technology with GEH., (Copyright 2017 PBJ-Associação Porto Biomedical/Porto Biomedical Society.)

Rapid cancer cell proliferation promotes the production of reducing equivalents, which counteract the effects of relatively high levels of reactive oxygen species. Reactive oxygen species levels increase in response to chemotherapy and cell death, whereas an increase in antioxidant capacity can confer resistance to chemotherapy and is associated with an aggressive tumor phenotype. The pentose phosphate pathway is a major site of NADPH production in the cell, which is used to maintain the main intracellular antioxidant, glutathione, in its reduced state. Previous studies have shown that the rate of hyperpolarized [1- 13 C]dehydroascorbic acid (DHA) reduction, which can be measured in vivo using non-invasive 13 C magnetic resonance spectroscopic imaging, is increased in tumors and that this is correlated with the levels of reduced glutathione. We show here that the rate of hyperpolarized [1- 13 C]DHA reduction is increased in tumors that have been oxidatively prestressed by depleting the glutathione pool by buthionine sulfoximine treatment. This increase was associated with a corresponding increase in pentose phosphate pathway flux, assessed using 13 C-labeled glucose, and an increase in glutaredoxin activity, which catalyzes the glutathione-dependent reduction of DHA. These results show that the rate of DHA reduction depends not only on the level of reduced glutathione, but also on the rate of NADPH production, contradicting the conclusions of some previous studies. Hyperpolarized [1- 13 C]DHA can be used, therefore, to assess the capacity of tumor cells to resist oxidative stress in vivo However, DHA administration resulted in transient respiratory arrest and cardiac depression, which may prevent translation to the clinic., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Non-invasive imaging of gene expression can be used to track implanted cells in vivo but often requires the addition of an exogenous contrast agent that may have limited tissue access. We show that the urea transporter (UT-B) can be used as a gene reporter, where reporter expression is detected using 1 H MRI measurements of UT-B-mediated increases in plasma membrane water exchange. HEK cells transfected with the reporter showed an increased apparent water exchange rate (AXR), which increased in line with UT-B expression. AXR values measured in vivo, in UT-B-expressing HEK cell xenografts, were significantly higher (about twofold, P < 0.0001), compared with non-expressing controls. Fluorescence imaging of a red fluorescent protein (mStrawberry), co-expressed with UT-B showed that UT-B expression correlated in a linear fashion with AXR. Transduction of rat brain cells in situ with a lentiviral vector expressing UT-B resulted in about a twofold increase in AXR at the site of virus injection., Competing Interests: The authors declare no competing financial interests.

Background: Recent studies have shown that 3'-deoxy-3'-[(18)F] fluorothymidine ([(18)F]FLT)) uptake depends on endogenous tumour thymidine concentration. The purpose of this study was to investigate tumour thymidine concentrations and whether they correlated with [(18)F]FLT uptake across a broad spectrum of murine cancer models. A modified liquid chromatography-mass spectrometry (LC-MS/MS) method was used to determine endogenous thymidine concentrations in plasma and tissues of tumour-bearing and non-tumour bearing mice and rats. Thymidine concentrations were determined in 22 tumour models, including xenografts, syngeneic and spontaneous tumours, from six research centres, and a subset was compared for [(18)F]FLT uptake, described by the maximum and mean tumour-to-liver uptake ratio (TTL) and SUV., Results: The LC-MS/MS method used to measure thymidine in plasma and tissue was modified to improve sensitivity and reproducibility. Thymidine concentrations determined in the plasma of 7 murine strains and one rat strain were between 0.61 ± 0.12 μM and 2.04 ± 0.64 μM, while the concentrations in 22 tumour models ranged from 0.54 ± 0.17 μM to 20.65 ± 3.65 μM. TTL at 60 min after [(18)F]FLT injection, determined in 14 of the 22 tumour models, ranged from 1.07 ± 0.16 to 5.22 ± 0.83 for the maximum and 0.67 ± 0.17 to 2.10 ± 0.18 for the mean uptake. TTL did not correlate with tumour thymidine concentrations., Conclusions: Endogenous tumour thymidine concentrations alone are not predictive of [(18)F]FLT uptake in murine cancer models.

Changes in tumor metabolism may accompany disease progression and can occur following treatment, often before there are changes in tumor size. We focus here on imaging methods that can be used to image various aspects of tumor metabolism, with an emphasis on methods that can be used for tumor grading, assessing disease progression, and monitoring treatment response. Clin Cancer Res; 22(21); 5196-203. ©2016 AACR., (©2016 American Association for Cancer Research.)